An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12

Abstract

The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes -the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although, no proteolytic activity has been described for these members. The goal of this present study was to investigate (1) if the ancient bovine PAGs (PAGs-2 and -12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAGs-2 and -12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k cat /K M for bovine PAG-2 was 2.7×10 5 M −1 s −1 and for boPAG-12 it was 6.8×10 4 M −1 s −1 . The enzymes were inhibited by pepstatin A with a K i of 0.56 and 7.5 nM for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.

Placental PAGs: gene origins, expression patterns, and use as markers of pregnancy

“…However, the majority of PAGs contain conserved features typically found in functional aspartic proteinases. In fact, proteolytic activity has been confirmed for a few of them (Telugu & Green 2008, Telugu et al 2010. The extent to which the proteolytic activity of PAGs is relevant to their function during pregnancy is unclear, but some speculation about how such activity could reflect their role(s) in pregnancy is discussed later in this review.…”

“…For example, Wooding et al (2005) noted that some PAGs, such as bovine PAG2 and porcine PAG2, accumulate extensively at the placenta-uterine interface. Both of these PAGs are known to possess proteolytic activity (Telugu & Green 2008, Telugu et al 2010. It seems possible that proteolytically active PAGs present at the interface could process latent growth factors that are known to be present at that location (Munger et al 1998, Rifkin et al 1999, Moussad et al 2002.…”

“…Protein binding via the cleft may be possible even for those PAGs that possess proteolytic activity. Similar to most aspartic proteinases, those PAGs that can act as proteinases have maximum activity at relatively low pH (Telugu & Green 2008, Telugu et al 2010. If the pH microenvironment is not low enough to promote proteolytic activity, the substrate-binding cleft could still serve as a docking site for other proteins.…”

Identification of embryonic/fetal mortality in cows by semiquantitative detection of pregnancy-associated glycoproteins

“…Recently, a positive correlation between PAG11 expression and prostaglandin-endoperoxidase synthase 2 (R¼ 0.87) and prostaglandin E synthase (R¼0.76), which are two key enzymes involved in prostaglandin E2 synthesis, has been reported (Thompson et al, 2012). Additionally, pHdependent proteolytic activities of PAG-2 and -12 have been shown in vitro (Telugu et al, 2010). It can be hypothesized that pathological uterine changes lead to a decline in pH, inducing proteolysis by PAGs, thereby causing placental separation.…”

Biology of the Placental Proteins in Domestic Ruminants: Expression, Proposed Roles and Practical Applications

“…Recent papers have reported that some ancient PAGs exhibit proteolytic activities as aspartic proteinase 61,62 . These ancient PAGs might act as a secreted aspartic proteinase during gestation, while cathepsins are localized in the cell.…”